| RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells
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Author:
Date:
2017-04-20
[Abstract] The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3’-5’ exoribonuclease. The following protocol describes an approach to monitor the ribonucleolytic activity of affinity-purified EXOSC10-containing RNA exosomes, originating from HEK-293 cells, as reported in (Domanski et al., 2016) and further detailed in the ...
[摘要] RNA外植体复合物在RNA加工和调节营养中起核心作用。在细胞质和细胞核中存在，外来体通过与核糖核酸酶和各种衔接蛋白的关联起作用（参见[Kilchert等人，2016]）。 RNA外植体相关的EXOSC10蛋白是分布的3'-5'外核糖核酸酶。以下方案描述了监测源自HEK-293细胞的亲和纯化含EXOSC10的RNA外来体的核糖核酸裂解活性的方法，如（Domanski等人，2016）所报道的，并进一步详细描述这个配套生物协议（Domanski和LaCava，2017）。
在我们以前的工作中，我们在四环素诱导型CMV启动子（HEK-293 Flp-In T-REx-Thermo）的控制下，建立了表达C末端3xFLAG标记的外来体成分EXOSC10（RRP6）的同基因HEK-293细胞系费雪科学）。该系统允许我们以与内源WT蛋白质相当的水平表达标记的EXOSC10蛋白质，并且使用磁性抗FLAG亲和介质和来源于冷冻细胞粉末的蛋白质提取物来研究外来体纯化方案（Domanski等， / ...
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| Measurement of Net High-affinity Urea Uptake in Maize Plants
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Author:
Date:
2015-06-05
[Abstract] Despite its extensive use as a nitrogen fertilizer, the role of urea as a directly accessible nitrogen source for crop plants is still poorly understood. So far, the physiological and molecular aspects of urea acquisition have been investigated only in a few plant species highlighting the importance of a urea transporter in roots, DUR3 (Kojima et al., 2007; Wang et al., 2012; Zanin et al., 2014a). Regarding maize plants, a crop that needs a large amount of urea fertilizer, the capability to take up urea via an inducible and high-affinity transport system has been ...
[摘要] 尽管其作为氮肥的广泛使用，但是作为直接可及的作物植物氮源的脲的作用仍然知之甚少。 迄今为止，仅在少数植物物种中研究了尿素获取的生理学和分子学方面，突出了根中的尿素转运蛋白DUR3的重要性（Kojima等人，2007; et al。，2012; Zanin et al。，2014a）。 关于玉米植物，需要大量尿素肥料的作物，通过诱导型和高亲和力转运系统摄取尿素的能力最近已被表征（Zanin等人，2014a; Zanin et al。，2014b）。 在这里，我们描述了一个小规模的协议，适合测量尿素净高亲和力摄取完好的玉米植物的根。
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| In vitro Transcription (IVT) and tRNA Binding Assay
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Author:
Date:
2014-09-20
[Abstract] This protocol describes the coupling of (i) “live” in vitro RNA transcription with (ii) binding by a radiolabeled, pre-formed tRNA followed by native gel electrophoresis and phosphorimager scan to visualize the complex. The necessity arose from the stable structure that one RNA forms in the absence of its interaction partner. The T-box leader RNA, a transcription control system, folds into a thermodynamically very stable stem-loop structure without the tRNA present, which makes in vitro binding interaction of both pre-formed RNAs very difficult. I therefore adjusted the ...
[摘要] 该方案描述了（i）"活"体外RNA转录与（ii）通过放射性标记的预先形成的tRNA的结合，然后是天然凝胶电泳和磷光成像仪扫描以显现复合物的偶联。 必要性来自一种RNA在不存在其相互作用配偶体时形成的稳定结构。 T盒前导RNA，转录控制系统，折叠成热力学非常稳定的茎 - 环结构，没有tRNA存在，这使得体外结合两个预先形成的RNA的相互作用非常困难。 因此，我调整结合测定以模拟细菌细胞中的"天然"情况，其中预先形成的稳定的tRNA已经存在，而T盒前导RNA被RNA聚合酶主动转录。 方案的第一部分还描述了体外转录和tRNA的标记。
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